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Replicative senescence is accompanied by a telomere‐specific DNA damage response (DDR). We found that DDR+ telomeres occur spontaneously in early‐passage normal human cells and increase in number with increasing cumulative cell divisions. DDR+ telomeres at replicative senescence retain TRF2 and RAP1 proteins, are not associated with end‐to‐end fusions and mostly result from strand‐independent, postreplicative dysfunction. On the basis of the calculated number of DDR+ telomeres in G1‐phase cells just before senescence and after bypassing senescence by inactivation of wild‐type p53 function, we conclude that the accrual of five telomeres in G1 that are DDR+ but nonfusogenic is associated with p53‐dependent senescence. Replicative senescence is triggered by DNA damage response foci associated with telomeres. Reddel and colleagues now establish that a threshold of five damaged telomeres exists to induce senescence in normal cells and that end‐to‐end chromosome fusion is not required for senescence induction.

Telomeric DNA is protected by the shelterin complex, whose disruption triggers DNA-damage responses, checkpoint activation and chromosomal fusions. Now analysis of human cell lines reveals a spontaneously occurring intermediate state in which the DNA-damage response is activated at the telomeres without cell cycle arrest or chromosomal fusions, and with TRF2 playing a central role in determining such a state. Telomere dysfunction is typically studied under conditions in which a component of the six-subunit shelterin complex that protects chromosome ends is disrupted. The nature of spontaneous telomere dysfunction is less well understood. Here we report that immortalized human cell lines lacking wild-type p53 function spontaneously show many telomeres with a DNA damage response (DDR), commonly affecting only one sister chromatid and not associated with increased chromosome end-joining. DDR + telomeres represent an intermediate configuration between the fully capped and uncapped (fusogenic) states. In telomerase activity–positive (TA + ) cells, DDR is associated with low TA and short telomeres. In cells using the alternative lengthening of telomeres mechanism (ALT + ), DDR is partly independent of telomere length, mostly affects leading strand–replicated telomeres, and can be partly suppressed by TRF2 overexpression. In ALT + (but not TA + ) cells, DDR + telomeres preferentially associate with large foci of extrachromosomal telomeric DNA and recombination proteins. DDR + telomeres therefore arise through different mechanisms in TA + and ALT + cells and have different consequences.

Ultrafast electron diffraction techniques that employ relativistic electrons as a probe have been in the spotlight as a key technology for visualizing structural dynamics which take place on a time scale of a few femtoseconds to hundreds femtoseconds. These applications highly demand not only extreme beam quality in 6-D phase space such as a few nanometer transverse emittances and femtosecond duration but also equivalent beam stability. Although these utmost requirements have been demonstrated by a compact setup with a high-gradient electron gun with state-of-the-art laser technologies, this approach is fundamentally restricted by its nature for compressing the electrons in a short distance by a ballistic bunching method. Here, we propose a new methodology that pushes the limit of timing jitter beyond the state-of-the-art by utilizing consecutive RF cavities. This layout already exists in reality for energy recovery linear accelerator demonstrators. Furthermore, the demonstrators are able to provide MHz repetition rates, which are out of reach for most conventional high-gradient electron guns.

Telomere lengths are maintained in many cancer cells by the ribonucleoprotein enzyme telomerase but can be further elongated by increasing telomerase activity through the overexpression of telomerase components. We report here that increased telomerase activity results in increased telomere length that eventually reaches a plateau, accompanied by the generation of telomere length heterogeneity and the accumulation of extrachromosomal telomeric repeat DNA, principally in the form of telomeric circles (t‐circles). Telomeric DNA was observed in promyelocytic leukemia bodies, but no intertelomeric copying or telomere exchange events were identified, and there was no increase in telomere dysfunction‐induced foci. These data indicate that human cells possess a mechanism to negatively regulate telomere length by trimming telomeric DNA from the chromosome ends, most likely by t‐loop resolution to form t‐circles. Additionally, these results indicate that some phenotypic characteristics attributed to alternative lengthening of telomeres (ALT) result from increased mean telomere length, rather than from the ALT mechanism itself.

Some immortalized mammalian cell lines and tumors maintain or increase the overall length of their telomeres in the absence of telomerase activity by one or more mechanisms referred to as alternative lengthening of telomeres (ALT). Characteristics of human ALT cells include great heterogeneity of telomere size (ranging from undetectable to abnormally long) within individual cells, and ALT-associated PML bodies (APBs) that contain extrachromosomal telomeric DNA, telomere-specific binding proteins, and proteins involved in DNA recombination and replication. Activation of ALT during immortalization involves recessive mutations in genes that are as yet unidentified. Repressors of ALT activity are present in normal cells and some telomerase-positive cells. Telomere length dynamics in ALT cells suggest a recombinational mechanism. Inter-telomeric copying occurs, consistent with a mechanism in which single-stranded DNA at one telomere terminus invades another telomere and uses it as a copy template resulting in net increase in telomeric sequence. It is possible that t-loops, linear and/or circular extrachromosomal telomeric DNA, and the proteins found in APBs, may be involved in the mechanism. ALT and telomerase activity can co-exist within cultured cells, and within tumors. The existence of ALT adds some complexity to proposed uses of telomere-related parameters in cancer diagnosis and prognosis, and poses challenges for the design of anticancer therapeutics designed to inhibit telomere maintenance.

Telomeres of eukaryotic chromosomes contain many tandem repeats of a G-rich sequence (for example, TTAGGG in vertebrates 1 ). In most normal human cells, telomeres shorten with each cell division, and it is proposed that this limits the number of times these cells can replicate 2 . Telomeres may be maintained in germline cells, and in many immortalized cells and cancers, by the telomerase holoenzyme 3 (first discovered in the ciliate Tetrahymena 4 ), which uses an RNA subunit as template for synthesis of telomeric DNA by the reverse transcriptase catalytic subunit 5 . Some immortalized human cell lines and some tumours maintain their telomeres in the absence of any detectable telomerase activity by a mechanism referred to as alternative lengthening of telomeres 6 , 7 (ALT). Here we show that DNA sequences are copied from telomere to telomere in an immortalized human ALT cell line, indicating that ALT occurs by means of homologous recombination and copy switching.

Telomeres, specialised structures that protect chromosome ends, play a critical role in preserving chromosome integrity. Telomere dynamics in the Tasmanian devil (Sarcophilus harrisii) are of particular interest in light of the emergence of devil facial tumour disease (DFTD), a transmissible malignancy that causes rapid mortality and threatens the species with extinction. We used fluorescent in situ hybridisation to investigate telomere length in DFTD cells, in healthy Tasmanian devils and in four closely related marsupial species. Here we report that animals in the Order Dasyuromorphia have chromosomes characterised by striking telomere length dimorphism between homologues. Findings in sex chromosomes suggest that telomere length dimorphism may be regulated by events in the parental germlines. Long telomeres on the Y chromosome imply that telomere lengthening occurs during spermatogenesis, whereas telomere diminution occurs during oogenesis. Although found in several somatic cell tissue types, telomere length dimorphism was not found in DFTD cancer cells, which are characterised by uniformly short telomeres. This is, to our knowledge, the first report of naturally occurring telomere length dimorphism in any species and suggests a novel strategy of telomere length control. Comparative studies in five distantly related marsupials and a monotreme indicate that telomere dimorphism evolved at least 50 million years ago.

Activation of a telomere maintenance mechanism seems to be indispensable for the immortalization of human cells. Most cancers and cancer cell lines maintain their telomeres via telomerase. In some cancers, however, telomeres are maintained in the absence of telomerase activity by one or more mechanisms that are known as alternative lengthening of telomeres (ALT). Successful telomere-targeted anticancer therapy might therefore require a combination of telomerase and ALT inhibitors, emphasizing the importance of understanding the molecular details of telomere maintenance mechanisms in immortal cells and their repression in normal cells.

We directly compared two methods of immortalizing human mammary epithelial cells (HMECs). Cells were transfected with an expression plasmid either for hTERT, the catalytic subunit of telomerase, or for the simian virus 40 (SV40) early region genes. Under standard culture conditions, HMECs were not immortalized by hTERT unless they had spontaneously ceased expression of the p16(INK4a) tumor suppressor gene. Untransfected HMECs had low levels of telomerase expression, and immortalization by both methods was associated with an increase in telomerase activity and prevention of telomere shortening. SV40-induced immortalization was accompanied by aberrant differentiation, loss of DNA damage response, karyotypic instability and, in some cases, tumorigenicity. hTERT-immortalized cells had fewer karyotypic changes, but had intact DNA damage responses, and features of normal differentiation. Although SV40-immortalized cells are useful for studies of carcinogenesis, hTERT-immortalized cells retain more properties of normal cells.

Exogenous expression of the catalytic subunit of telomerase, hTERT, in a normal human foreskin fibroblast cell strain resulted in telomerase activity and an extended proliferative lifespan prior to a period of crisis. Three immortalized cell lines with stably maintained telomere lengths were established from cells that escaped crisis. Each of these cultures underwent a significant downregulation of p16(INK4a) expression due to gene deletion events. One cell line also acquired mutations in both alleles of the p53 tumor suppressor gene. Downregulation of p16(INK4a) and loss of wild-type p53 expression occurred after escape from crisis, so these mutations are most likely not required for immortalization of these cells but rather were selected for during continuous growth in vitro. These findings emphasize the need for caution in the use of hTERT-immortalized cells in studies of normal cell biology or in tissue engineering and the need to monitor for genetic instability and the accumulation of mutations in both the p16(INK4a)/pRb and p53 pathways.